Biosearch Technologies

20 November 2008

A Match Made In Heaven
Multiplexing with CAL Fluor®, Quasar®
and Black Hole Quencher® Dyes.

Multiplexing real-time, quantitative PCR assays using unoptimized reporter dye sets places unusual performance and data analysis demands on today's real-time, qPCR thermocyclers.

Biosearch has developed two series of performance-optimized dyes for multiplex real-time, quantitative PCR-the CAL Fluor® and Quasar® Dyes. Having emission maxima ranging from 522 nm to 670 nm, these new dyes are high performing, low cost alternative dyes for seven fluorophores commonly used on real-time thermocyclers: TET, JOE, VIC, HEX, Texas Red®, Cy™3 and Cy5.

Coupled with our BHQ® dark quenchers you now can realize the full potential of your qPCR thermocycler!
*Please note: Probe offer limited to one per customer; for domestic orders only; 25 nmol scale orders excluded.

Old Dye	New Dye	ABS	EM	Quencher
TET	CAL Fluor® Gold 540	522	544	BHQ®-1
VIC/HEX/JOE	CAL Fluor Orange 560	538	559	BHQ-1
Cy™3	Quasar 570	548	566	BHQ-2
TAMRA	CAL Fluor Red 590	569	591	BHQ-2
Texas Red	CAL Fluor Red 610	590	610	BHQ-2
LC Red 640	CAL Fluor Red 635	618	637	BHQ-2
Cy5	Quasar 670	647	667	BHQ-2
Cy5.5	Quasar 705	690	705	BHQ-2

All Major Thermocyclers and All Probe/Primer Formats

The CAL Fluor, Quasar and BHQ dyes can be incorporated into a variety of popular probe designs, including dual-labeled TaqMan® and Molecular Beacon probes and Black Hole Scorpions™ and Amplifluor® Direct primer systems. And, they are fully compatible with the range of real-time PCR instruments including the Applied Biosystems' and Stratagene real-time machines, the Corbett Rotor-Gene™ 6000, the Bio-Rad iQ5® and Cepheid SmartCycler®, among others.

Duplex, Triplex and even Quadraplex Capability

Use this spectral overlay chart to view our multiplexing recommendations, compare spectra, and pair dyes with Black Hole Quenchers.

Multiplexing Recommendations¹ for Dual-Labeled Dark-quenched Probes and Primers
Machine	Company	Calibration Required?²	Multiplexing Degree	Dye 1	Dye 2	Dye 3	Dye 4	Dye 5
Prism® 7700	ABI	Yes	Duplex	FAM	CAL Fluor® Gold 540	SuperROX®³
Prism 7900	ABI	Yes	Duplex	FAM	CAL Fluor® Gold 540	SuperROX
Prism 7000	ABI	Yes	Duplex	FAM	CAL Fluor® Gold 540	SuperROX
Prism 7300	ABI	Yes	Duplex	FAM	CAL Fluor® Orange 560	SuperROX
Prism 7500	ABI	Yes	Quadraplex	FAM	CAL Fluor® Orange 560	TAMRA	SuperROX	Quasar® 670
iQ®	Bio-Rad	Yes	Quadraplex	FAM	CAL Fluor® Orange 560	CAL Fluor® Red 610	Quasar® 670
iQ 5	Bio-Rad	Yes	Pentaplex	FAM	CAL Fluor® Gold 540	CAL Fluor® Red 590	CAL Fluor® Red 610	Quasar® 670
SmartCycler®	Cepheid	Yes	Triplex	FAM	CAL Fluor® Orange 560	CAL Fluor® Red 635
SmartCycler II	Cepheid	Yes	Quadraplex	FAM	CAL Fluor® Orange 560	CAL Fluor® Red 610	Quasar® 670
Rotor-Gene™ 3000	Corbett Research	No	Quadraplex	FAM	CAL Fluor® Orange 560	CAL Fluor® Red 610	Quasar® 670
Rotor-Gene 6000	Corbett Research	No	Pentaplex	FAM	CAL Fluor® Gold 540	CAL Fluor® Red 610	Quasar® 670	Quasar® 705
Mastercycler® ep Realplex	Eppendorf	Yes	Duplex	FAM	CAL Fluor® Gold 540
Opticon® 2	MJ Research/ Bio-Rad	Yes	Duplex	FAM	CAL Fluor® Orange 560
Chromo™ 4	MJ Research/ Bio-Rad	Yes	Quadraplex	FAM	CAL Fluor® Orange 560	CAL Fluor® Red 610	Quasar® 670
MX3000P™	Stratagene	No	Quadraplex	FAM	CAL Fluor® Orange 560	CAL Fluor® Red 610	Quasar® 670
MX4000®	Stratagene	No	Quadraplex	FAM	CAL Fluor® Orange 560	CAL Fluor® Red 610	Quasar® 670
LightCycler® 1.2	Roche	Yes⁴	Duplex	FAM	Pulsar® 650
LightCycler 2.0	Roche	Yes⁴	Triplex	FAM	CAL Fluor® Red 610	Pulsar 650
LightCycler 480	Roche	Yes⁴	Quadraplex	FAM	CAL Fluor® Orange 560	CAL Fluor® Red 610	Quasar® 670
Determined through research at Biosearch Determined through research of a collaborator Predicted, based on instrument specifications

Click here for an electronic copy of our most recent Dye Selection and Multiplex Recommendations Chart.

An additional real and measurable advantage in using the CAL Fluor and Quasar dyes lies in multiplex qPCR. Their pairing with Biosearch's proprietary BHQ dark quenchers in dual-labeled probes enables not only duplex but also triplex and quadraplex qPCR capability.

Dual-labeled probes incorporating CAL Fluor or Quasar dyes coupled with a BHQ dye exhibit large signal to noise values and produce amplification traces with robust ΔRns and earlier CT values. This capability was powerfully demonstrated in a poster presented by Biosearch at the recent CHI meeting "PCR and Beyond 2004" in McLean, Virginia, where real-time data showing the simultaneous amplification of four different genomic DNA targets in a quadraplex assay was presented

(click to enlarge)

These traces illustrate the performance of the CAL Fluor and Quasar dyes in both single and quadraplex reactions, amplifying targets from the human genes Alpha Tubulin, Interleukin 1 beta, GAPDH, and Factor VIII. The CT value for each amplified target in singleplex reactions (colored traces) is nearly equivalent to that target when amplified in multiplex reactions (black traces).

Lower Dual-Labeled Probe Cost

Instrument Compatibility
(Click to see PDF file)

Dual-labeled probes represent a significant cost investment of qPCR. And, if you're considering duplex, triplex and even quadraplex qPCR, your probe costs grow significantly.

Dual-labeled probes synthesized with JOE, VIC and Texas Red (only available as esters whose use is labor intensive), TET and HEX (which suffer from instability during post DNA synthesis work-up), and Cy3 and Cy5 (which are expensive dyes) require careful and experienced handling during synthesis and purification to guarantee probes of outstanding quality.

The CAL Fluor and Quasar dyes overcome each of these disadvantages: probe synthesis with the CAL Fluor and Quasar dyes can be automated, they are extremely stable to DNA probe work-up conditions and this translates into cost savings to you, the scientist.

These recommendations apply to dual-labeled BHQ-quenched probes (TaqMan® Probes, Molecular Beacons) and BHQ-quenched primers (Black Hole Scorpions, Amplifluor Direct, Plexor, etc). They should not be used as guidelines for TAMRA-quenched probes or for Hybridization Probes that rely on FRET as a means of excitation. Recommendations highlighted in blue have not been proven and so should be used cautiously on an experimental basis. Stratagene customers select their filters from among a series upon instrument purchase. Because multiplexing dye compatibility is affected by filter specifications, the above Stratagene recommendations only apply to the standard FAM/HEX/ROX/Cy5 filter set.
Some instruments will require fluorescence calibration. Fluorescence calibration allows these instruments to record the spectral profile for the dyes(s) to be used in subsequent assays by resolving the total detected light into signals contributed by the individual fluorophores. Please visit our calibration dye page for additional information.
SuperRox is a proprietary passive reference available from Biosearch.
LightCycler users can calibrate using TaqMan® probes directly and therefore aren't required to purchase dye calibration kits to perform color compensation.